More on SEM, signal detection sample prep Flashcards
Everhardt-Thornley Detector (ETD)
SEM Sensor for secondary electrons, or potentially low energy backscattered electrons. A positively charged grid attracts these electrons towards a scintillator. The scintillator produces a luminescent output when hit by electrons. This light signal is carried by a light pipe to be further amplified by a photomultiplier.
Principle of BSE detector
Solid state (photodiode) detector
they are often CBS: Concentric Back-Scattered
segmented detectors. THeir distribution is either only concentric so it allows for angle senstivity of the BSEs or the concentric segments are split further allowing for directional detection as well. (see figure if still confused).
In addition, BSE detectors can either be located at different places, to distinguish further traveling BSEs (or oven front scattered ones for STEM)
Where can you have BSE detectors?
In-Column Detectors,
Mirror Detectors
Through-the-Lens detectors
Standard/Below obejctive above sample
Under sample (for STEM)
Describe one of the most common ways to disentangle the BSE and SE signals.
Immersion
Applying an electric field pole piece and sample (stage bias) will create a retarding electric field that will pull SEs astray more effectively than BSE as BSE have higher energy. Thus you would get a clearer signal.
This works differently if you have magnetic lenses. Then the magnetic field already pulls out the SEs to the central axis. However, due to the spiraling, a lot of BSEs are lost. Here immersion actually allows to detect more BSEs as you can attract them to the detector.
*Immersion is created by charging negatively your sample stage
What will happen to a sample if it is non-conductive / not grounded?
It will get charged. For low E0 (beam energy) they will be negatively charged. Increading E0 till a certain point decreases charging effect untill the charging becomes positive. This is because now electrons are being elastically scattered while also inelastically pushing away electrons making the sample lose negative charges. However, this effect reaches a maximum at a certain point and begins to shift back to negative charging as the amount of applied electrons in combination with SEs being reverted to the sample , overcomes the lost ones. Thus there are two possible spots where charging effect is diminished.
In addition, charged artifacts / contaminants like dust can also influence this. They can create small local electric fields because of which dark spots. That would be the case for negatively charged dust particles. It will induce positive charges in the sample which would then pull in electrons -> dark spots.
A thin biological tissue section can best be imaged using
A. Secondary electron detector
B. Back-scattered electron detector
C. Transmission electron detector
Explanation:
Transmission Electron Detector (C): In a transmission electron microscope (TEM) setup, electrons pass through the thin sample and are collected by a detector on the other side. This mode provides high-resolution details of the internal structure of thin samples, making it ideal for imaging biological tissue sections, which are typically only tens to hundreds of nanometers thick.
Secondary Electron Detector (A): Secondary electron detectors are primarily used in standard SEM to capture surface information and topography. However, they do not provide sufficient internal structural information, especially for thin, transparent biological samples, as they detect low-energy electrons ejected from near the surface.
Back-Scattered Electron Detector (B): While backscattered electron detectors in SEM offer contrast based on atomic number, they are less effective for ultra-thin samples where internal structural details are desired. Biological tissues generally have low atomic number contrast, making BSE less informative for this purpose
Some general consideration in sample prep for SEM
- vacuum: your sample should be fixed: using aldehydes most often,
- dehydration: again distortion of the sample and thus fixation is necessary
- if you want to see the internal structures of a cell: sectioning is crucial. Mandatory for STEM
- contrast: chemically through heavy metals or if phase contrast, freezing is needed (but thats tem)
Which of these statements is NOT true?
Increasing the electron beam current:
A. Allows to reduce the pixel dwell time
B. May lead to resolution loss due to aberrations
and Coulomb interaction
C. Is done by selecting a larger beam energy
D. Is done by selecting a larger aperture
C. Increasing the electron beam current is done by selecting a larger beam energy.
Explanation:
(A) Allows to reduce the pixel dwell time: This is true. Increasing the beam current provides a higher electron flux, allowing more signal to be collected in less time per pixel, thereby reducing the pixel dwell time required for imaging.
(B) May lead to resolution loss due to aberrations and Coulomb interaction: This is also true. Higher beam current can cause increased electron-electron (Coulomb) interactions and more significant lens aberrations, both of which can degrade the resolution of the image.
(C) Is done by selecting a larger beam energy: This is NOT true. Increasing the beam energy (accelerating voltage) does not directly increase the current. Beam energy refers to the speed and penetration power of the electrons rather than the number of electrons (current). To increase current, other adjustments such as changing the condenser lens settings or using a larger aperture are used.
(D) Is done by selecting a larger aperture: This is true. Using a larger condenser aperture allows more electrons to pass through, effectively increasing the beam current.
General fixation approaches
- Chemical: aldehydes; osmium tetroxide
- Cryo: vitrification, no ice formation. Water expands &
‘purifies’ upon freezing (phase separation)
plunge freezing or high-pressure freezing (HPF)
How to preserve structure when sectioning?
Embedding the sample in resin beforehand. Then thin cuts with a microtome will distort less the sample. Also makes it practical to actually section it.
Some standard ways of labeling for EM
- Nanoparticles immunolabeling: Au (15 nm) & Quantum dots. The Antiobodies bind to protein, but this process is tricky for fixed embedded samples. Then NPs are very well visible in EM
- miniSOG (mini singlet oxygen generator): endogeneously expressed fluorescent probe but also generates ROS that result in oxidation of DAB(added externally). It polymerizes. This aggregated structure binds more to contrast agents like Osmium oxide and makes the sample more visible on EM.
A well-established SEM-CLEM method for (neural) tissue imaging.
Array tomography.
ribbons of serial ultrathin sections representing substantial tissue volumes can be cut and collected on a single glass slide and simultaneously processed and imaged for the reconstruction of the three-dimensional distribution of antigens. Staining antibodies can be subsequently eluted and the sections restained a number of times thus allowing the detection of tens of antigens in the same sample. Furthermore, planarization of the three-dimensional specimen completely eliminates problems of both staining and imaging efficiency depending upon depth within a tissue that have previously prevented quantitative interpretation of the resulting volume images
